Compositions and methods for producing and using homogenous neuronal cell transplants

ABSTRACT

Methods of treating individuals suspected of suffering from diseases, conditions or disorders of the Central Nervous System which comprise implanting stable, homogeneous post-mitotic human neurons into the individual&#39;s brain are disclosed. Methods of treating individuals suspected of suffering from injuries, diseases, conditions or disorders characterized by nerve damage which comprise implanting stable, homogeneous post-mitotic human neurons at or near a site of said nerve damage. Pharmaceutical compositions comprising stable, homogeneous post-mitotic human neurons and a pharmaceutically acceptable medium are disclosed. Methods of generating non-human animal models of human CNS diseases, conditions or disorders which comprise implanting stable, homogeneous post-mitotic human neurons into the brain of a non-human animal are disclosed. Non-human animals comprising stable, homogeneous post-mitotic human neurons implanted in their brain are disclosed.

[0001] This invention was made in the course of research sponsored bythe NIH grant number NS18616. The United States Government has certainrights in this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions useful for andmethods of transplanting stable, homogeneous populations of neuron cellsinto non-human animals in order to generate non-human animal modelsuseful to study human diseases, conditions and disorders. The presentinvention relates to compositions useful for and methods oftransplanting stable, homogeneous populations of neuron cells intoindividuals in order to treat or prevent diseases, conditions anddisorders, especially those characterized by loss, damage or dysfunctionof the brain and/or loss, damage or dysfunction of an individualsneurons at other sites in the individual's body. This application isrelated to U.S. patent application Ser. No. 09/122,019 filed Jul. 24,1998, U.S. patent application Ser. No. 08/150,368 filed Nov. 9, 1993,U.S. patent application Ser. No. 08/170,668 filed Dec. 17, 1993, U.S.patent application Ser. No. 07/911,980 filed Jul. 10, 1992, and U.S.patent application Ser. No. 07/780,715 filed Oct. 21, 1991, now U.S.Pat. No. 5,175,103 issued Dec. 29, 1992, which are each incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0003] The transplantation of major categories of central nervous system(CNS) cells (i.e. neurons, astrocytes) or CNS tissue fragments offersopportunities to study the developmental biology and immunologicalproperties of these cells, to create animal models of CNS diseases suchas Alzheimer's disease and to develop alternative strategies for thetreatment of relentlessly progressive neurodegenerative disorders suchas Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis and hereditary ataxia as well as to studyother diseases, conditions and disorders characterized by loss, damageor dysfunction of neurons including transplantation of neuron cells intoindividuals to treat individuals suspected of suffering from suchdiseases, conditions and disorders. Indeed, recent pioneering efforts toutilize human fetal mesencephalic tissue grafts to ameliorate theextrapyramidal manifestations of drug induced and idiopathic Parkinson'sdisease emphasize the potential of transplanted human CNS tissues forthe treatment of human neurodegenerative diseases (Freed, C. A., et al.1992 New Engl. J. Med. 327:1549-1555; Spencer, D. D. et al. 1992 NewEngl. J. Med. 327:1541-1548; and Widner, H., et al. 1992 New Engl. J.Med. 327:1556-1563). However, the results of these efforts have not beencompletely satisfactory.

[0004] The immortalization of CNS progenitor cells using constructscontaining temperature sensitive promoters has enabled transplantationof genetically engineered precursors of neurons and glia, but braingrafts of these progenitors have given rise to mixed populations ofglial and neuronal progeny (Cattaneo, E., and R. McKay 1991 TINS14:338-340; Renfranz, P. J., et al. 1991 Cell 66:713-729; Snyder, E. Y.,et al. 1992 Cell 68:33-51). An alternative strategy has been to useneuron-like transformed cell lines obtained from tumors of the CNS, butneoplastic neuron-like cells usually cannot be induced to permanentlyexit the cell cycle or they develop into tumors when transplanted intothe rodent brain (Fung, K.-F. et al. 1992 J. Histochem. Cytochem.40:1319-1328; Trojanowski, J. Q., et al. 1992 Molec. Chem. Neuropathol.17:121-135; and Wiestler, O. D. et al. 1992 Brain Pathol. 2:47-59). Aslowly dividing human neuronal cell line obtained from a child withunilateral megalencephaly was shown to exhibit a neuron-like phenotypein culture but grafts of these cells in the rodent CNS showed a mixtureof neuronal and mesenchymal phenotypic properties (Poltorak, M., et al.1992 Cell Transplant I:3-15).

[0005] There is a need for a method of generating animal models of CNSdiseases and disorders by transplanting neurons into the brains of suchanimals to produce conditions which resemble or mimic CNS diseases,conditions or disorders.

[0006] There is a need for animal models of CNS diseases and disordersby transplanting neurons into the brains of such animals to produceconditions which resemble or mimic CNS diseases, conditions ordisorders.

[0007] There is a need for a method of treating individuals suspected ofsuffering from CNS diseases, conditions or disorders by transplantingneurons in order to replace or introduce cells whose presence reversesor impedes the pathology associated with the disease being treated.

[0008] There is a need for a method of treating individuals suspected ofsuffering from neuron damage caused by stroke or injury such as headtrauma, nerve injury or spinal injury by transplanting neurons in orderto replace cells damaged by stroke or an injury.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a method of treating anindividual suspected of suffering from a disease, condition or disordercharacterized by the damage or loss of neurons which comprisesimplanting a sample from a culture of at least 95% pure, stable,homogeneous post-mitotic human neurons into the individual at or nearthe site of the damage or loss.

[0010] The present invention relates to a method of treating anindividual suspected of suffering from an injury, disease, condition ordisorder of the Central Nervous System which comprises implanting asample from a culture of at least 95% pure, stable, homogeneouspost-mitotic human neurons into the individual's brain.

[0011] The present invention relates to a method of treating anindividual suspected of suffering from an injury, disease, condition ordisorder to the spinal cord which comprises implanting a sample from aculture of at least 95% pure, stable, homogeneous post-mitotic humanneurons into the individual's spinal column.

[0012] The present invention relates to a method of treating anindividual suspected of suffering from an injury, disease, condition ordisorder to nerve cells which comprises implanting a sample from aculture of at least 95% pure, stable, homogeneous post-mitotic humanneurons into the individual's body at the site of nerve dysfunction ordamage.

[0013] The present invention relates to a pharmaceutical compositionthat comprises a sample from a culture of at least 95% pure, stable,homogeneous post-mitotic human neurons and a pharmaceutically acceptablemedium.

[0014] The present invention relates to a method of generating anon-human animal model for a human disease, condition or disorder of theCentral Nervous System comprising implanting a sample from a culture ofat least 95% pure, stable, homogeneous post-mitotic human neurons into anon-human animal.

[0015] The present invention relates to an non-human animal comprising asample from a culture of at least 95% pure, stable, homogeneouspost-mitotic human neurons implanted in its brain, nervous system orspinal column.

DESCRIPTION OF DRAWINGS

[0016]FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G andFIG. 1H contain photomicrographs of NT2N graft in the hippocampus(dentate gyrus and polymorph layer) 4 weeks post-transplant probed withvarious monoclonal antibodies.

[0017]FIG. 2A, FIG. 2B and FIG. 2D show photomicrographs of threedifferent NT2N grafts in the subcortical white matter and the dorsaldiencephalon (FIG. 2C) 2-4 weeks post-transplant stained with CresylViolet (FIG. 2A, FIG. 2C and FIG. 2D) or the MAb (ED1) to macrophages(FIG. 2B).

[0018]FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G andFIG. 3H contain photomicrographs of an NT2N graft in the subcorticalwhite matter at 4 weeks post-transplant probed with MAbs andcounterstained with hematoxylin.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to compositions and methodsrelating to transplanting neurons into either individuals who aresuspected of suffering from an injury, disease, disorder or condition orinto non-human animals to generate a non-human animal model of a humandisease, disorder or condition. The neurons used in the methods of thepresent invention are at least 95% pure, stable, homogeneouspost-mitotic human neurons. Optionally, the neurons may be compriseexogenous genetic material. The neurons used in the methods of thepresent invention are genotypically and phenotypically homogenous.

[0020] As used herein, the term “sample” is meant to refer to one ormore cells. In preferred embodiments, a sample contains a plurality ofcells. According to the present invention, a sample from a culture of atleast 95% pure, stable, homogeneous post-mitotic human neurons isimplanted into either a non-human animal or a human. Accordingly, themethods of the present invention relate to the implantation of one ormore cells from a culture of at least 95% pure, stable, homogeneouspost-mitotic human neurons into either a non-human animal or a human.

[0021] As used herein, the term “at or near a site of said nerve damage”is meant to refer to the location where nerve cells are implanted inorder to replace destroyed, damaged or dysfunctional nerve cells and/orrestore function resulting from destroyed, damaged or dysfunctionalnerve cells. The location is defined as being a site where suchimplanted cells can develop as replacement cells for destroyed, damagedor dysfunctional nerve cells and make the necessary linkages to restorefunction lost due to destroyed, damaged or dysfunctional nerve cells.

[0022] As used herein, the term “exogenous genetic material” refers togenomic DNA, cDNA, synthetic DNA and RNA, mRNA and antisense DNA and RNAwhich is introduced into the cell or an ancestor cell. The exogenousgenetic material may be heterologous or an additional copy or copies ofgenetic material normally found in the individual or animal. When cellsare used as a component of a pharmaceutical composition in a method fortreating human injuries, diseases, conditions or disorders, theexogenous genetic material that is used to transform the cells mayencode proteins selected as therapeutics used to treat the individualand/or to make the cells more amenable to transplantation. When cellsare used in a method for generating non-human animal models of human CNSdiseases or disorders, the exogenous genetic material that may beincorporated into the cells may encode proteins selected to createconditions in the non-human animal which simulates or resemblesconditions in a human suffering from CNS disease, condition or disorderto be modeled.

[0023] The exogenous genetic material is preferably provided in anexpression vector which includes the coding region of a protein whoseproduction by the cells is desirous operably linked to essentialregulatory sequences such that when the vector is transfected into thecell, the exogenous genetic material is capable of being expressedwithin the cell.

[0024] According to some embodiments of the present invention, a samplefrom a culture of pure, stable, homogeneous post-mitotic human neuronsis transplanted into an individual being treated for a CNS injury,disease, condition or disorder. These cells essentially replace and/orfunction in place of endogenous damaged, dead, non-functioning ordysfunctioning cells. Thus, in the case of an individual suffering froman injury, disease, condition or disorder characterized by loss, damageor dysfunction of neurons such as, for example, diseases associated withnerve damage or spinal injury, the cells are transplanted into a site inthe individual where the transplanted cells can function in place of thelost, damaged or dysfunctional cells and/or produce products needed toimprove or restore normal functions that have been reduced or lost dueto the lack of such products endogenously produced in the individual. Inthe case of an individual suffering from a CNS injury, disease,condition or disorder characterized by loss, damage or dysfunction ofneurons in the brain, the cells are transplanted into the brain of theindividual. The transplanted cells function in place of the lost,damaged or dysfunctional cells and/or produce products needed to improveor restore normal brain functions that have been reduced or lost due tothe lack of such products endogenously produced in the individual.

[0025] According to some embodiments of the present invention, a samplefrom a culture of pure, stable, homogeneous post-mitotic human neuronsare transplanted into the individual being treated for a disease,condition or disorder in order to provide living neurons which producedesired substances. The transplanted cells may produce specific productsthat, when present at or near the site of implantation in the treatedindividual, reverses or impedes the pathology associated with thedisease, condition or disorder being treated.

[0026] According to some embodiments of the present invention, a samplefrom a culture of pure, stable, homogeneous post-mitotic human neuronsare transplanted into a non-human animal in order to provide a model fora human CNS disease, condition or disorder. The transplanted cells mayproduce products that result in the development of conditions which aresimilar to or mimic the pathology of a CNS disease or condition.

[0027] The method may be used to treat individuals suffering frominjuries, diseases, conditions or disorders characterized by the loss,damage or dysfunction of endogenous cells. The method may be used totreat individuals suffering from stroke, spinal injury or otherinjuries, conditions or disorders associated with neuron damage ordeath. CNS diseases and disorders which may be treated by practicing themethods of the present invention include any disease of the CNS which ischaracterized by the loss, damage or dysfunction of endogenous cells,the symptoms of which may be reversed or reduced in severity byproviding neurons that can replace such cells and produce productsneeded for proper function or needed to counteract the presence ofcompounds that are not normally present or present at abnormal levels.The present invention is useful for the treatment of progressiveneurodegenerative disorders such as Alzheimer's disease, Parkinson'sdisease, Huntington's disease, amyotrophic lateral sclerosis andhereditary ataxia as well as neurological conditions such as strokes andnerve injuries. The present invention is useful to treat diseases byserving as a delivery system to produce and disseminate active proteinsand other active compounds needed for proper brain function.

[0028] A pharmaceutical composition according to the present inventionuseful for treating individuals suffering from injuries, diseases,conditions or disorders characterized by the loss, damage or dysfunctionof endogenous cells comprises a sample from a culture of pure, stable,homogeneous post-mitotic human neurons and a pharmaceutically acceptablemedium. The neurons used in the present invention must be a stable,homogeneous culture of post-mitotic human neurons that is at least 95%pure. The neurons used in the present invention may be transfected withexogenous genetic material.

[0029] The exogenous genetic material used to transform the cells mayencode proteins selected as therapeutics for delivery to the brain ofthe treated individual. Protein products encoded by transfected geneticmaterial include, but are not limited to, those leading to production ofneurotransmitters (e.g. tyrosine hydroxylase) as well as neurotrophicsubstances such as nerve growth factor (NGF), brain-derived neurotrophicfactor (BDGF), basic fibroblast growth factor (bFGF) and glial-derivedgrowth factor (GDGF). In addition, tumor suppressor genes such as P53and thrombospondin can be incorporated into the cells.

[0030] According to another embodiment of the present invention, asample from a culture of pure, stable, homogeneous post-mitotic humanneurons are transplanted into the brain of a non-human animal in orderto generate a non-human animal model of a human CNS disease, conditionor disorder. The presence of the cells bring about changes in theanimal's brain such that animal develops features which resemble ormimic the characteristics of the human CNS diseases, conditions ordisorders. The transplanted cells produce specific products that, whenpresent in the brain of the animal, give rise to conditions whichresemble or mimic the pathology associated with the disease beingmodeled. The cells used to generate the non-human animal modelsaccording to the present invention useful for treating CNS diseasescomprises a sample from a culture of pure, stable, homogeneouspost-mitotic human neurons and a pharmaceutically acceptable medium. Theneurons used in the present invention must be a stable, homogeneousculture of post-mitotic human neurons that is at least 95% pure.

[0031] CNS diseases and disorders which may be modeled by practicing themethods of the present invention include any disease of the CNS which ischaracterized by endogenous dead, non-functioning or dysfunctioningcells, particularly those characterized by cells producing proteins notnormally associated with the cells or producing normal proteins atabnormal levels. Thus, the transplantation into the brain of an animalof cell which produce proteins associated with a human CNS disease givesrise to conditions which resemble or mimic the characteristicsassociated with the pathology of the disease or disorder being modeled.The present invention is useful to generate non-human animal models ofprogressive neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, hereditary ataxia, and motor neuron and Lewy body disease.Many different genes are implicated in these diseases such as normal andmutated amyloid precursor genes, genes encoding kinases, phosphotases,normal and mutated superoxide dismutase, neurofilament proteins andapolipoprotein 4. In addition, specific oncogenes responsible forcertain types of cancer can be incorporated to generate animal modelsfor such cancer using the NT2 derived cells.

[0032] In some embodiments of the present invention, the neurons usedmay be produced by a method described in U.S. Pat. No. 5,175,103 issuedDec. 29, 1992, which teaches a method for obtaining >95% purepostmitotic human neurons (termed NT2N cells) from a humanteratocarcinoma cell line (termed NTera2/clone PI or NT2 cells);following treatment of the NT2 cells with retinoic acid (RA). Inaddition to providing a model system for a wide range of biochemical,molecular biological and morphological studies of neurons in vitro, thestable, homogeneous population of pure human neurons may be used in invivo transplants in order to generate animal models of CNS diseases anddisorders or they may be used in in vivo transplants into the brains ofindividuals suffering from CNS diseases or disorders astherapeutics/prophylactics to introduce neurons which are capable ofproducing products that reverse or impede the pathology associated withCNS diseases or disorders afflicting the individual.

[0033] The NT2 cell line is unique among all other teratocarcinoma celllines that are capable of differentiating into neurons, glia andmesenchymal cells, because the NT2 cells appear to correspond toprogenitor cells, the progeny of which are restricted to the neuronallineage (Abramham, I. et al. 1991 J. Neurosci. Res. 28:29-39; Andrews,P. W., et al. 1981 Tissue Antigens 17:493-500; Andrews, P. W. et al1984. 1984 Lab. Invest. 50 147-162; Andrews, P. W. 1987. Devel. Biol.103:285-293; Kleppner, S. R., et al 1992 Soc. Neurosci. Abst. 18:782;Lee, V. M.-Y. and P. W. Andrews 1986 J. Neurosci. 6:514-521; and,Younkin, D. P. et al. 1993 Proc. Natl. Acad. Sci. U.S.A. 90:2174-2178).Further characterization of the NT2N cells has shown that these cellsmost closely resemble CNS neurons (Pleasure, S. J., and V. M. -Y. Lee.1993 J. Neurosci. Res. In press; and Pleasure, S. J., et al. 1992. J.Neurosci. 12:1802-1815). The NT2N cells exhibit other interestingproperties of CNS neurons, i.e. they express almost exclusively the 695amino acid long amyloid precursor protein (APP), produce and secrete theβ-amyloid or A4 (β/A4) peptide found in Alzheimer's disease amyloidplaques and bear glutamate receptor channels on their cell surface.

[0034] The neurons used in the present invention may be transfected withexogenous genetic material. If produced as described in U.S. Pat. No.5,175,103, the neurons used in the present invention may be transfectedwith genetic material prior to induction of differentiation. Methods oftransfection are well known and taught in the above-referenced patent.The exogenous genetic material used to transform the cells may encodeproteins whose presence within cell of the brain are associated withhuman diseases, disorders or conditions. Protein products encoded bytransfected genetic material include, but are not limited to, normal andmutated amyloid precursor, kinases, phosphotases, normal and mutatedsuperoxide dismutase, neurofilament proteins and apolipoprotein 4 aswell as neurotransmitters (e.g., tyrosine hydroxylase) and neurotrophicsubstances such as nerve growth factor (NGF), brain-derived neurotrophicfactor (BDGF), basic fibroblast growth factor (bFGF) and glial-derivedgrowth factor (GDGF).

[0035] The exogenous genetic material used to transfect the cells ispreferably provided in a vector which includes essential regulatorysequences operably linked to coding sequences such that the transfectedgenetic material is capable of being expressed within the cell.

[0036] Expression vectors that encode exogenous genetic materialcomprise a nucleotide sequence that encodes a protein to be producedoperably linked to regulatory elements needed for gene expression.Accordingly, incorporation of the DNA or RNA molecule into the neuroncell results in the expression of the DNA or RNA encoding the proteinand thus, production of the protein.

[0037] The exogenous genetic material that includes the nucleotidesequence encoding the protein operably linked to the regulatory elementsmay remain present in the cell as a functioning episomal molecule or itmay integrate into the cell's chromosomal DNA. Exogenous geneticmaterial may be introduced into cells where it remains as separategenetic material in the form of a plasmid. Alternatively, linear DNAwhich can integrate into the chromosome may be introduced into the cell.When introducing DNA into the cell, reagents which promote DNAintegration into chromosomes may be added. DNA sequences which areuseful to promote integration may also be included in the DNA molecule.Alternatively, RNA may be introduced into the cell.

[0038] The necessary elements of an expression vector include anucleotide sequence that encodes a protein and the regulatory elementsnecessary for expression of that sequence in the cells. The regulatoryelements are operably linked to the nucleotide sequence that encodes theprotein to enable expression. The nucleotide sequence that encodes theprotein may be cDNA, genomic DNA, synthesized DNA or a hybrid thereof oran RNA molecule such as mRNA.

[0039] The regulatory elements necessary for gene expression include: apromoter, an initiation codon, a stop codon, and a polyadenylationsignal. It is necessary that these elements be operable in the neurons.Moreover, it is necessary that these elements be operably linked to thenucleotide sequence that encodes the protein such that the nucleotidesequence can be expressed in the neuron cells and thus the protein canbe produced.

[0040] Initiation codons and stop codon are generally considered to bepart of a nucleotide sequence that encodes the protein. However, it isnecessary that these elements are functional in the neurons.

[0041] Similarly, promoters and polyadenylation signals used must befunctional within the neuron cells.

[0042] Examples of promoters useful to practice the present inventioninclude but are not limited to cytomegalovirus promoter, particular theimmediate early promoter, SV40 promoter and retroviral promoters.

[0043] An examples of polyadenylation signals useful to practice thepresent invention includes but is not limited to SV40 polyadenylationsignal.

[0044] An additional element may be added which serves as a target forcell destruction if it is desirable to eliminate transplanted cells forany reason. An expressible form of a herpes thymidine kinase (tk) genecan be included in the exogenous genetic material. When the exogenousgenetic material is introduced into the neuron, tk will be produced. Ifit is desirable or necessary to kill the transplanted cells, the druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk. Thus, a system canbe provided which allows for the selective destruction of transplantedcells.

[0045] In order for exogenous genetic material in an expression vectorto be expressed, the regulatory elements must be operably linked to thenucleotide sequence that encodes the protein. Accordingly, it isnecessary for the promoter and polyadenylation signal to be in framewith the coding sequence. In order to maximize protein production,regulatory sequences may be selected which are well suited for geneexpression in the neuronal cells. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce exogenous genetic material as expression vectorswhich are functional in neurons.

[0046] Neurons may be transplanted into individuals suspected ofsuffering from injuries, diseases, conditions or disorders characterizedby the damage or loss of neurons at the site of such neuron injury orloss by direct grafting of neurons at the site of neuron injury or loss.Neurons may be transplanted into the brains of individuals suspected ofsuffering from CNS diseases, conditions or disorders by direct graftingof neurons into the brains of such individuals. Additionally, neuronsmay be transplanted into the brains of individuals suffering from headtrauma or strokes. Individuals suspected of or identified as sufferingfrom diseases, conditions, disorders, or injuries rendering neurons inthe brain damaged, destroyed or dysfunctional may be treated byimplantation of neurons to replace or compensate for the loss of neuronfunction due to the destruction or dysfunction of endogenous neurons.

[0047] In some embodiments, 1×10³ to 1×10⁶ neurons are implanted. Insome embodiments, 5-10×10⁴ neurons are implanted. Two techniques havebeen used for neural transplantation, the first comprises stereotaxicsurgery in which a neuron cell suspension is implanted into the brain,the second in which the cells are grafted into the brain bymicrosurgery. Techniques for transplanting neural tissue are disclosedin: Backlund, E.-O. et al. , (1985) J. Neurosurg. 62:169-173; Lindvall,O. et al. (1987) Ann. Neurol. 22:457-468; and Madrazo, I. et al. (1987)New Engl. J. Med. 316:831-834; each of which is incorporated herein byreference.

[0048] Neurons may be implanted into the spinal cord at or near the siteof nerve damage from disease or injury. The implanted cells furtherdifferentiate into motor neurons, thereby replacing or reconnectingnerves at the site of damage. In some embodiments, the injury is to amotor neuron which is part of the spinal cord. In some embodiments, theinjury is to a motor neuron outside the spinal column. Neurons of theinvention are implanted at the site of the nerve cell injury, i.e., inproximity to the injured cell or cells at a location wheredifferentiation of implanted cells can replace nerve function andreconnect nerves of the individual to remedy or otherwise ameliorate theinjury. The neurons are implanted in a location that allows processeswhich develop therefrom to substitute for the processes of the damagednerve, thereby repairing the damaged nerve network.

[0049] Neurons may be transplanted into the brains of non-human animalsby injection of neurons into one hemisphere using a stereotaxicinstrument and a hand-held 10 ml Hamilton syringe. Aliquots of 1×10³ to1×10⁶ neurons are injected into the adult and neonatal rats. In someembodiments, 5-10×10⁴ neurons are injected. For the adult rats, cellsare injected stereotaxically into cerebral cortex, subjacent whitematter or hippocampus at one site in a single hemisphere of each rat.

[0050] The present invention is further illustrated by the followingexamples, which are not intended to be limiting in any way.

EXAMPLES Example 1

[0051] Studies of NT2N cells transplanted into the brains of nude miceshow that these NT2N cells integrate as neurons into the brains ofimmunodeficient nude mice where they survive >12 months without evidenceof rejection or tumor formation. Furthermore, the transplantation ofNT2N neurons in cyclosporine-treated and untreated immunocompetentSprague-Dawley rats has been performed and survival of the cells hasbeen observed.

[0052] The following is a review of experiments demonstratingimplantation of transfected neuronal cells into immunocompetent animals.

Materials and Methods

[0053] Culture of NT2 cells and generation of NT2N neurons wereperformed essentially as described in U.S. Pat. No. 5,175,103. Briefly,NT2 cells were cultured using standard techniques and were passaged 1:3twice per week in OptiMEM with 5% fetal bovine serum andpenicillin/streptomycin. NT2 cells were induced to differentiate intoneurons by administration of 10 μm retinoic acid (RA), which wasreplenished twice weekly, for 5 weeks at which time the cells werereplated to establish Replate 1 cells. Highly differentiated NT2N cellswere then obtained following two subsequent replate manipulations(designated Replate 2 and Replate 3) at which time the NT2N cellswere >99% pure. Freshly harvested aliquots of Replate 3 NT2N neuronswere washed three times in buffer and then used in the transplantationstudies described here.

[0054] Additionally, in experiments conducted in 2 rats, previouslyfrozen aliquots of NT2N cells were thawed immediately prior to injectioninto the CNS.

Implantation of NT2N Cells Into Rat Brain

[0055] Adult (170-280 gm) female Sprague/Dawley rats were anesthetizedby intraperitoneal injections of Ketamine (87 mg/kg) and Xylazine (13mg/kg), prepared for surgery and placed in a stereotaxic instrument(Kopf, Tujunga, Calif.). Neonatal (postnatal day 5) femaleSprague/Dawley rats were anesthetized by hypothermia during theinjection of NT2N cells into one hemisphere using a stereotaxicinstrument and a hand-held 10 μl Hamilton syringe. Aliquots of 5-10×10⁴NT2N cells were injected into the adult and neonatal rats. For the adultrats, NT2N cells were injected stereotaxically into cerebral cortex,subjacent white matter or hippocampus at one site in a single hemisphereof each rat. A total of 68 rats were used in this study (see Table 1).

[0056] The stereotaxic injection sites were determined using system B ofPellegrino et al. (Pellegrino, L. J., et al. 1979. A Stereotaxic AtlasOf The Rat Brain, Plenum Press, New York) and all of the injections wereperformed by injecting 2 μl of a suspension of the NT2N cells over 5min. After the injection, the needle was left in place for another 5min. and then slowly removed. The viability of the NT2N cells beforethey were injected was monitored microscopically using Trypan blueexclusion. Similar procedures were used to monitor the viability ofresidual, uninjected NT2N cells after the transplantation procedure hadbeen completed.

[0057] A subset of the adult rats (N=13) implanted with NT2N cells weretreated daily by the oral (N=8; using a gauvage tube) or subcutaneous(N=5) administration of cyclosporine (7-10 mg/kg per day) for theduration of their survival post-transplantation.

[0058] Following different post-transplantation survival times, the ratswere deeply anesthetized and sacrificed by perfusion with phosphatebuffered saline (to wash out red blood cells and serum proteins)followed by 70% ethanol and 150 mM NaCl. The brains were removed andfixed by overnight immersion in 70% ethanol and 150 mM NaCl. Thepost-transplant survival times ranged from 4 days to 21 weeks assummarized in Table 1.

[0059] Table 1 summarizes data on the number of adult (with and withoutsubcutaneous or oral cyclosporine treatment) and neonatal rats used fortransplantation as well as the survival times post-transplantation foreach group of rats (left and middle columns). The number of rats withviable NT2N grafts is shown in the far right column. The number of ratstreated with subcutaneous (sc) cyclosporine is indicated in parentheses.

Immunohistochemical Procedures

[0060] The methods for tissue processing and light microscopicimmunohisto-chemical analysis are well known. Antibodies were used forthe immunohistochemical characterization of the NT2N grafts. Bothmonoclonal and polyclonal antibodies to neuronal and glial cytoskeletalproteins and other polypeptides that have been shown to serve asmolecular signatures of the neuronal or glial phenotype were selected toidentify and characterize the NT2N grafts. These antibodies have beenextensively characterized and their properties are summarized in Table2.

[0061] Specifically, Table 2 summarizes the properties of the 27different antibodies used in this study and their reactivity with NT2Ncells grafted into the rat brain. The far left column indicates thepolypeptide recognized by the antibody which is named in the secondcolumn. The third column gives the dilution or immunoglobulinconcentration of each antibody as it was applied here. The fourth columnindicates whether or not the antibody stained grafted NT2N cells (+positive; −—negative; +/−—weak or equivocal staining). The antibodiesare grouped together according to the cell types in which they arepredominantly or exclusively expressed.

[0062] The abbreviations used in the first column of the Table 2 (inalphabetical order) are:

[0063] GFAP=Glial fibrillary acid protein;

[0064] MAP2=Microtubule-associated protein 2;

[0065] MAPS=Microtubule-associated protein 5;

[0066] MBP=myelin basic protein;

[0067] N-CAM=Neural-cell adhesion molecule;

[0068] NF=Neurofilament;

[0069] NF-L=Low molecular weight NF protein;

[0070] NF-M=Middle molecular weight NF protein;

[0071] NF-H=High molecular weight NF protein;

[0072] p75 NGFR=Low affinity (75 kD) nerve growth factor receptor;

[0073] P^(ind)=Phosphate independent epitope in NF-L or NF-H;

[0074] P⁻=Non- or poorly phosphorylated epitope in NF-H or NF-M;

[0075] P⁺=Moderately phosphorylated epitope in NF-H;

[0076] P⁺⁺⁺=Heavily phosphorylated epitope in NF-H;

[0077] PHF=Paired helical filaments in Alzheimer's diseaseneurofibrillary tangles.

[0078] Note that two antibodies (i.e., T3P and PHF1) to tau proteinsrecognize fetal tau and the abnormally phosphorylated tau proteins (atserine number 396 according to the numbering system for the 441 aminoacid long tau protein) in PHFs (also known as A68 proteins), but notnormal adult tau. Note that although the anti-CFAP and anti-macrophageMAbs stained occasional reactive astrocytes and macrophages,respectively, that had infiltrated the graft, the NT2N cells themselveswere not stained by these MAbs.

RESULTS Specific Identification of NT2N Grafts with MonoclonalAntibodies

[0079] FIG. a and FIG. 1B contains photomicrographs of NT2N graft in thehippocampus (dentate gyrus and polymorph layer) 4 weeks post-transplant.FIG. 1A shows a low power view of a Cresyl Violet stained section of theNT2N graft (delineated by the arrows). FIG. 1B shows a low power view ofthe same NT2N graft stained with the human specific anti-N-CAM MAb (MOC1). The asterisk lies above the portion of the graft containing theperikarya and simple dendritic arbor of the NT2N neurons while the arrowheads identify the axons emanating from the graft and extending in themossy fiber pathway dorsal to pyramidal neurons in CA3. The regionidentified by the asterisk is shown at higher power in FIG. 1C and thesegment of graft-derived axons located below the middle arrow head isshown at higher power in FIG. 1D. Note that only the NT2N neurons andtheir processes are stained by this MAb. FIG. 1A and FIG. 1B are at thesame magnification and the bar in FIG. a=100 μm. FIG. 1C and FIG. 1Dshow a higher power views of the NT2N graft stained by the humanspecific anti-N-CAM MAb (MOCl). Note that the NT2N neurons and some oftheir dendrites (FIG. 1C) as well as their axons (FIG. 1D) are stained,but not the endogenous rodent N-CAMs. The photomicrographs in FIG. 1C,FIG. 1F, FIG. 1G and FIG. 1H are all at the same magnification and thebar in FIG. 1C=100 μm, while FIG. 1D and FIG. 1E are taken at a slightlyhigher magnification and the bar in FIG. 1C corresponds to 25 μm in FIG.1D and FIG. 1E. FIG. 1E and FIG. 1F show regions similar to thoseillustrated in FIG. 1C and FIG. 1D, respectively, in an adjacent sectionstained with the MAb RHdO20 (FIG. 1D) to poorly phosphorylated NF-H/Mand the MAb HO14 (FIG. 1F) to moderately phosphorylated isoforms ofNF-H. FIG. 1G shows results obtained with a MAb to highly phosphorylatedNF-H (RMO24) which stains only endogenous rat axons, but not the NT2Ngraft (arrows) despite the fact that RMO24 also recognizes human NF-H.The section shown in FIG. 1H is adjacent to that seen in FIG. 1F and itwas probed with the MAb to GFAP. Some reactive astrocytes infiltrate thegraft similar to the colonization of dorsal root ganglion graftstransplanted into rat brain, but most CFAP positive reactive astrocytessurround the graft. The sections in FIG. 1B-FIG. 1H were lightlycounterstained with hematoxylin.

[0080]FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D show photomicrographs ofthree different NT2N grafts in the subcortical white matter (FIG. 2A,FIG. 2B and FIG. 2D) and the dorsal diencephalon (FIG. 2C) 2-4 weekspost-transplant stained with Cresyl Violet (FIG. 2A, FIG. 2C and FIG.2D) or the MAb (ED1) to macrophages (FIG. 2B). FIG. 2A and FIG. 2B areadjacent sections of the same graft and the arrow heads identify theinterface between the graft (above) and the subjacent white matter(below). FIG. 2A and FIG. 2B are at the same magnification and the barin FIG. 2A=50 μm. The arrows in FIG. 2B identify ED1 positivemacrophages in an area of the graft containing some NT2N neuronsundergoing focal karyorrhexis. More extensive inflammation is seenaround blood vessels in FIG. 2C (arrow) at the margin of the graft(star) while more severe karyorrhexis of grafted NT2N cells is seen inanother subcortical white matter NT2N graft shown in FIG. 2D where thearrows identify accumulations of nuclear debris. FIG. 2C and FIG. 2D areat different magnifications and the bar in FIG. 2A corresponds to 100 μmin FIG. 2C and to 30 μm in FIG. 2B.

[0081]FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G andFIG. 3H contain photomicrographs of an NT2N graft in the subcorticalwhite matter at 4 weeks post-transplant probed with MAbs andcounterstained with hematoxylin. The section shown in FIG. 3A wasstained with the human specific anti-NF-H MAb (H014) which demonstratesthe grafted perikarya and their dendrites in the NT2N transplant to theright in the figure. Labeled axons extend medially from the graft siteto the left in this panel. Note that these axons cross the midline(star) within the corpus callosum. The section in FIG. 3B, which was asection adjacent to that shown in FIG. 3A, was probed with a MAb tohuman N-CAM (MOC1) which stains the somatodendritic domain of NT2N cellsin the graft to the right in this figure as well as axons that cross themidline (star) to the left within the corpus callosum. FIG. 3A and FIG.3B are at the same magnification and the bar in FIG. 3A=100 μm. Theaxons in the corpus callosum seen in FIG. 3A and FIG. 3B are shown athigher power in FIG. 3C and FIG. 3D, respectively. These axons (arrowsin FIG. 3C and FIG. 3D) cross the midline (star in FIG. 3C and FIG. 3D)to the hemisphere contralateral to the NT2N graft. FIG. 3C and FIG. 3Dare at different magnifications and the bar in FIG. 3C=100 μm while thesame bar corresponds to 50 μm in FIG. 3D. In FIG. 3E, the MAb to MAP2(APl4) labels the somatodendritic domain of the grafted NT2N neurons.The cell body mass of the graft is identified by an asterisk and theoverlying white matter (WM) is unstained. The somatodendritic domain ofendogenous host neurons in the overlying cortex also are labeled in thissection and labeled apical dendrites are most prominent at thismagnification. FIG. 3E is at the same magnification as FIG. 3A and FIG.3B. In FIG. 3F, the MAb to highly phosphorylated NF-H (RHO24) does notstain the NT2N neurons and their processes in the graft (asterisk).However, endogenous axons in the surrounding white matter (WM) arelabeled by this MAb thereby delineating the extent of the cell body massand dendrites of this graft. The bar in FIG. 3F=100 μm. The twophotomicrographs shown in FIG. 3G and FIG. 3H are high power views ofthe NT2N grafts in adjacent sections stained with the anti-NF-Lantiserum (FIG. 3G) and the MAb (TA51) to moderately phosphorylatedisoforms of NF-H (FIG. 3H). Note that many of the NT2N neurons containimmunoreactive NF-L and NF-H (arrows in FIG. 3G and FIG. 3H,respectively) in their perikarya and processes. Additionally, endogenousrodent axons in the white matter (upper left in FIG. 3G and FIG. 3H)also are labeled by these antibodies. FIG. 3G and FIG. 3H are at thesame magnification and the bar in FIG. 3F corresponds to 50 μm in FIG.3G and FIG. 3H.

[0082] Although the grafts could be recognized in Cresyl Violet stainedsections (FIG. 1A, FIG. 2A, FIG. 2B and FIG. 2D), the identification oftransplanted NT2N cells in the rodent CNS was greatly facilitated byexploiting the restriction of certain polypeptides or epitopes containedwithin some of these polypeptides to human versus rat and mature versusimmature CNS neurons. For example, MOC1, the monoclonal antibody (MAb)to human neural cell adhesion molecules (N-CAMs), was shown to recognizeN-CAMs in the human NT2N neurons, but not the N-CAMs in the rat CNS(FIG. 1B-FIG. 1D). Indeed, the cytology of the NT2N cells was notsufficiently distinctive to allow recognition of the NT2N cells withoutthe use of immunohistochemistry. Furthermore, axons arising from theNT2N grafts were only identifiable as graft derived when they werelabeled with the human polypeptide specific antibodies described here(FIG. 1B, FIG. 1F, FIGS. 3A-3D). In addition to the anti-N-CAM MAb, thegrafted NT2N cells also could be specifically identified with the MAbH014, an antibody that recognizes moderately phosphorylated isoforms ofthe middle (NF-M) molecular weight (Mr) neurofilament (NF) subunit inthe human CNS and in NT2N cells, but not in the rodent CNS (FIG. 1F). Incontrast, RMO24 (FIG. 1G) and RMO217, both of which are MAbs to the mostheavily phosphorylated isoforms of the high (NF-H) Mr NF subunit thatappear only in mature CNS neurons, immunostained NF-H in rodent CNSneurons, but these MAbs did not stain the human NT2N cells in the graftsdescribed here. The inability of RMO24 and RM0217 to stain the graftedNT2N cells probably reflects the incomplete phosphorylation of NF-H inthe grafted NT2N cells (which reflects the incomplete maturation ofthese grafted neurons), since both MAbs recognize phosphorylated NF-Hextracted from the fully mature, human CNS. If the NT2N cells areallowed to survive for an extended period of time (i.e., >6 months) inthe immunodeficient nude mouse brain, then the grafted NT2N neuronsacquire the most heavily phosphorylated isoforms of NF-H and these fullymature grafted neurons are labeled by RMO24 and RMO217. However, graftedNT2N cells were only studied here for post-transplant survival times of<4 months, and both RMO24 and RMO217 strongly stained rat CNS neurons,but not the grafted NT2N cells, and MOC1 and HO14 stained the NT2Ngrafts specifically and intensely, but not rat CNS neurons or other ratCNS cells. Thus, all 4 of these MAbs were used to screen sections fromall 68 rats that received implants of the NT2N cells in order tospecifically identify the surviving NT2N grafts. Additionally, a MAb(2.2B10) to glial fibrillary acidic protein (GFAP) stained reactiveastrocytes surrounding the graft (FIG. 1H) which also helped to delimitthe NT2N grafts. Some of these reactive astrocytes infiltrated the NT2Ngrafts (FIG. 1H) similar to the colonization of dorsal root gangliongrafts by reactive astrocytes transplanted into the rat brain. Screeningthe graft sites with this panel of MAbs provided a highly effectivestrategy for identifying grafted NT2N cells even when they existed assmall clumps trapped in the leptomeninges or in the needle track dorsalto the injection site.

Survival of Grafted NT2N Cells

[0083] Nearly all of the transplanted NT2N cells were accuratelyimplanted into neocortex, subjacent white matter and hippocampusalthough a few also were detected in the diencephalon, the lateralventricle or within the leptomeninges overlying the neocorticalinjection site. The number and disposition of the grafted NT2N cellsvaried from rat to rat, but NT2N grafts were identifiedimmunohistochemically in 100% of adult (N=5) and neonatal (N=5) ratsthat survived for up to 2 weeks without cyclosporine treatment (seeTable 1 for a summary of these and the following data on NT2N graftsurvival). This group of rats with viable NT2N grafts included 2 ratstreated with cyclosporine that had been implanted with aliquots ofpreviously frozen NT2N cells. At the next post-transplant survivalinterval, i.e., 4 weeks, 10/24 adult and 2/2 neonatal rats that were nottreated with cyclosporine contained NT2N brain grafts (Table 1), andmany of the transplanted cells resembled small stellate neuronsmorphologically and histochemically in Niss1 stained preparations of thegrafts. However, at subsequent post-transplant survival times, only 1 of19 adult or neonatal rats that were not treated with cyclosporinecontained identifiable, surviving NT2N neurons. These findings reflectrejection of the NT2N grafts rather than the cessation of expression ofN-CAMs and NF proteins by the grafted NT2N cells. This conclusion isbased upon 4 reasons:

[0084] 1) inflammatory cells were detected in some of the viable graftsin association with cellular debris as early as 2 weekspost-transplantation (FIG. 2C) and many of these inflammatory cells wereidentified as macrophages using the macrophage specific ED1 MAb (FIG.2B);

[0085] 2) cyclosporine prolonged the survival of all NT2N grafts in ratsthat received this agent by a subcutaneous route;

[0086] 3) the maximum number of macrophages and inflammatory cells werenoted to infiltrate the graft site at 2-4 weeks post-transplantation;and

[0087] 4) in the immuno-deficient nude mouse, grafted NT2N cellssurvive >12 months, continue to express N-CAMs and NF proteins, andprogressively mature such that they acquire a fully mature neuronalphenotype by 12 months post-transplant.

[0088] Of the 5 rats that received subcutaneous cyclosporine, all 5contained viable NT2N grafts at post-transplant intervals that rangedfrom 2 to 12 weeks. In contrast, administration of cyclosporine bygauvage at the same dose (i.e., 7-10 mg/kg) appeared less effective inpreventing graft rejection since only 2 of 8 rats treated in this mannercontained an identifiable NT2N graft (Table 1). Notably, theadministration of cyclosporine to these rats did not appear to have anydetectable effect on the ability of the surviving NT2N cells to expressa range of neuronal polypeptides.

Maturation of Grafted NT2N Cells and the Establishment of NeuronalPolarity

[0089] Presumably, as a result of their progressive maturation in vivo,NT2N grafts that survived 2-4 weeks post-transplantation were thelargest and the most amenable to serial section immunohistochemicalanalysis, while only a limited number of sections containingidentifiable NT2N cells could be obtained from rats that survived 4 daysto 1 week post-transplantation. For this reason, studies were focused onthe maturational state and polarity of the NT2N cells on rats thatsurvived 2-4 weeks post-transplantation. At these time points, NT2Ncells in hippocampus or in the subcortical white matter (whichconsistently contained larger populations of NT2N cells than theneocortex perhaps due to leakage of the NT2N cells from the corticalinjection site into the overlying subarachnoid space) expressed severalwell characterized polypeptides (e.g., NF subunits and other neuronalcytoskeletal proteins, synaptic polypeptides) that unequivocallyidentified the NT2N cells as neurons (see FIGS. 3A-3E, FIG. 3G, FIG. 3Hand Table 2). However, these neurons resembled late fetal human spinalcord (i.e., >25 weeks gestational age) or young postnatal humancerebellar (i.e., <1 year old) neurons rather than fully mature neuronsof the adult CNS in that they failed to acquire heavily phosphorylatedisoforms of NF-H. In contrast, polypeptides expressed by glial cellswere infrequent in these grafts and the presence of rare GFAP positiveastrocytes in these grafts (FIG. 1H) undoubtedly reflects the migrationof reactive rat astrocytes into the grafts.

[0090] Four week old NT2N neurons extended axons over severalmillimeters (FIGS. 1B-1F), and some of these axons projected to thehemisphere contralateral to the graft site (FIGS. 3A-3D). Althoughdendrites were readily identified because they could be labeled withantibodies to microtubule associated proteins (MAPs) restricted to thesomatodendritic domain (e.g., MAP2), these dendrites were short with asimplified branching pattern (FIG. 3E). Nonetheless, the presence ofidentifiable axons and dendrites containing polypeptides that werecompartmentalized like their counterparts in authentic rat or humanneurons in vivo (FIGS. 1B-1F and FIGS. 3A-3E, FIG. 3G and FIG. 3H)indicate that by 4 weeks post-transplantation the grafted NT2N neuronshad acquired the molecular phenotype and structural polarity seen innearly mature human CNS neurons in vivo. Further, none of the graftedNT2N cells expressed proteins (e.g., nestin, vimentin, p75 NGFR) thatare found in neuronal progenitor cells or very immature (i.e.,“nascent”) human CNS neurons. Significantly, despite evidence ofneuronal degeneration due to graft rejection (FIGS. 2A-2D), none of thegrafts showed evidence of neuronal cytoskeletal protein abnormalitiessimilar to those seen in common neurodegenerative diseases. Finally,there was no evidence (e.g. mitoses, metastases) to indicate that any ofthe surviving NT2N cells reverted to a neoplastic phenotype.

DISCUSSION

[0091] This study demonstrates the properties of CNS transplants of purepopulations of clonal human neuron-like cells that are capable ofundergoing progressive, normal maturation and integration into the hostmammalian brain without evidence of tumor formation. Only one other CNScell line, the human HCN-1 line, appears to exhibit an exclusive invitro commitment to the neuronal lineage, but this cell line does notmaintain a stable neuronal phenotype when transplanted into the CNS ofexperimental animals (Ronnett, C. V. et al. 1990 Science 248:603-605.

[0092] The paucity of suitable neuronal cell lines for transplantationhas limited studies of the immunological response of the CNS totransplanted neurons alone. This report demonstrates that the NT2Nneurons are capable of expressing antigens that induce rejection byabout 4 weeks post-transplant. Although the precise nature of theseantigens in the NT2N cells is unknown at this time, humanteratocarcinoma cell lines similar to the NT2 parent cell line have beenshown to express major histocompatibility antigens such as HLA-A, B andC antigens and β₂ microglobulin.

[0093] More significantly, this study demonstrates that transplantedNT2N neurons are capable of undergoing partial neuronal maturation inthe rat brain. The NT2N cells injected into the rat brain progressivelymatured to about the same extent as their in vitro counterpartsmaintained in culture for up to 28 days following Replate 3. However,they did not attain the same level of maturity as transplanted NT2Ncells that survived for >9-12 months in the immunodeficient nude mousebrain. Specifically, the NT2N grafts in the rat brain did progress to alevel of maturation by 4 weeks post-transplant that corresponded to thematurational state of authentic human neurons in the late embryonicspinal cord or in the immature, young postnatal cerebellum. They differsignificantly from olfactory sensory neurons and the neuron-like tumorcells in CNS medulloblastomas. However, the transplanted and culturedNT2N cells do resemble the differentiated and fairly mature neurons thathave been observed in situ in some teratocarcinomas and many teratomas.

[0094] Based on the findings presented here, the transplantation of theNT2N cells into experimental animals can be exploited for several typesof unique studies of the developmental biology of neurons and theregressive neurodegenerative events that occur in some neurologicaldisorders. First, the ability to “re-start” the process of neuronalmaturation and the development of neuronal polarity by transplanting theNT2N cells into different regions of the rodent brain can be used asmodels of these two important developmentally regulated processes. Theavailability of an effective human model system to study these processesin a controlled experimental setting should greatly facilitate effortsto gain insights into the regulatory mechanisms that govern theseprocesses. This model system also will allow the opportunity to explorethe possibility that the micro-environment of the host brain mightinduce NT2N cells grafted into different neuroanatomical sites to assumea region specific morphological and neurotransmitter phenotype.

[0095] Moreover, wild type or genetically modified NT2N cells can beused to develop animal models of humans diseases, conditions anddisorders, particularly nervous system diseases. For example, the NT2Ncells preferentially express the 695 amino acid long Amyloid precursorprotein (APP₆₉₅) and they secrete the β/A4 peptide into the culturemedium. Hence, the wild type NT2N cells, NT2N cells transfected tooverexpress APP₆₉₅, or NT2N cells transfected to overexpress β/A4 can betransplanted in order to provide an animal model that releases APP₆₉₅ orβ/A4 into the extracellular space following transplantation. Thedeposition of βA4 peptides that occur in the Alzheimer's disease braincan be modeled in this way.

[0096] Transplantation of NT2N cells genetically engineered to producebioactive molecules can be used to develop novel methods to circumventthe blood-brain barrier for the treatment of human neurodegenerativediseases. For example, in view of the therapeutic promise evidenced byrecent studies of the use of fetal mesencephalic grafts for thetreatment of Parkinson's disease, induction of NT2N cells to acquire adopaminergic phenotype for use in the treatment of Parkinson's diseasefollowed by transplantation can be a therapy for individuals suspectedof suffering from Parkinson's disease.

Example 2 Transfection and Staining for β-galactosidase

[0097] Highly purified populations of neurons from a humanteratocarcinoma cell line were obtained as described in U.S. Pat. No.5,175,103. When undifferentiated, the NT2 cells were transfected with100 μg SPUD1 and 10 μg of pSV2neo by lipofection using LIPOFECTINtransfecting reagent (Bethesda Research Laboratories). SPUD1 is aβ-galactosidase expression vector which utilizes the SV40 promoter andhas Moloney murine leukemia virus long terminal repeats upstream anddownstream. After two days in complete medium, the transfectants wereselected with 200 μg/ml G418 (Gibco) for seven days. Cells were stainedfor β-galactosidase activity with 1 mg/ml X-gal, 5 mM potassiumferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl₂ in PBS afterfixation in 2% paraformaldehyde, 0.2% glutaraldehyde in phosphatebuffered saline pH 7.4. β-gal positive cultures were subcloned twice andthe subclones were used for further studies. The cells were photographedusing Hoffman modulation contrast to allow the simultaneousvisualization of the blue reaction product and the processes.

[0098] The β-galactosidase (β-gal) expression plasmid was shown to bepresent in both undifferentiated and post-mitotic cells. Thus,transfection of expression plasmids into undifferentiated cells allowsthe introduction of exogenous genetic material into cells. The cells canthen be induced to become stable, post-mitotic human neurons and canexpress the exogenous genetic material. TABLE 1 POST-TRANSPLANTATIONSURVIVAL DATA FOR GRAFTED NT2N CELLS Post- Number Of Rats Number Of Ratstransplant injected with viable survival w/NT2N Cells NT2N cell graftsADULT UNTREATED RATS  4 Days 3 3  2 Weeks 2 2  4 Weeks 24 10  6 Weeks 40  8 Weeks 3 1 13 Weeks 2 0 SUBTOTAL = 38 SUBTOTAL = 16 NEONATALUNTREATED RATS  1 Week 2 2  2 Weeks 3 3  4 Weeks 2 2  8 Weeks 2 0 12Weeks 2 0 16 Weeks 2 0 16 Weeks 2 0 21 Weeks 2 0 SUBTOTAL N = 17SUBTOTAL N = 7 ADULT CYCLOSPORINE TREATED RATS  2 Weeks 3 3 (sc = 1) (sc= 1)  4 Weeks 1 1 (sc)  6 Weeks 1 1 (sc)  8 Weeks 2 1 (sc = 1) (sc = 1)10 Weeks 2 0 11 Weeks 3 0 12 Weeks 1 1 (sc) SUBTOTAL = 13 SUBTOTAL = 7GRAND GRAND TOTAL = 68 TOTAL = 30

[0099] TABLE 2 POLYPEPTIDE AND CELL SPECIFICITY OF ANTIBODIES AND THEIRREACTIVITY WITH GRAFTED NT2N CELLS DILUTION NT2N POLYPEPTIDE ANTIBODYμg/ml GRAFT NEURONS Clathrin light chain LCB2 0.1 +/− MAP2 APl4 1:100 +MAP5 AA6 1:1500 + NF-H, P⁺⁺⁺ RMO24 Neat − NF-H, P⁺⁺⁺ RMO217 Neat − NF-H,P⁺ TA51 1:20 + NF-H/M, P⁻ RMdO20 1:10 + NF-H, P⁺⁺ HO14 1:25 + NF-M,P^(ind) RMO254 1:25 + NF-L, P^(ind) NR 4 1:10 + NF-L, P^(ind) Anti-NF-L1:50 + Neuron Specific Protein NST11 1:10 − Protein Kinase Cγ PKCGG Neat− Tau T14 Neat + Tau 134 1:500 +/− Tau (fetal/PHF) T3P 1:50 + Tau(fetal/PHF) PHF1 1:2000 +/− NEURONS AND NEUROENDOCRINE CELLSChromogranin LK2h10 1:500 − Synaptophysin SY 38 1:100 +/− TyrosineHydroxlase Anti-TH 1:100 − NEUROEPITHELIAL STEM CELLS Nestin Anti-Nestin1:2000 − GLIAL CELLS GFAP 2.2B10 1:500 − MBP Anti-MBP 1:100 − NEURAL,MESENCHYMAL & OTHER CELLS N-CAM MOC 1 1:10 +  p75 NGFR Me 20.4 1:100 −Vimentin V9 1:100 − Macrophage marker ED1 1:500 −

1. A method of treating an individual suspected of suffering from acentral nervous system disease or disorder comprising the step of:implanting a sample from a culture of at least 95% pure, stable,homogeneous post-mitotic human neurons into said individual's brain. 2.The method of claim 1 wherein said neurons are differentiated NT2Ncells.
 3. The method of claim 1 wherein said neurons are transfected,differentiated NT2N cells that have been transfected with exogenousgenetic material, wherein said transfected, differentiated cells expresscoding sequences of said exogenous genetic material to produce a proteinproduct.
 4. The method of claim 3 wherein said exogenous geneticmaterial includes nucleic acid sequences that encode proteins selectedfrom the group consisting of neurotransmitters and neurotrophicsubstances such as nerve growth factor, brain-derived neurotrophicfactor (BDGF), basic fibroblast growth factor (bFGF) and glial-derivedgrowth factor.
 5. The method of claim 1 wherein said central nervoussystem disease or disorder is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease, strokeand nerve injuries.
 6. A pharmaceutical composition comprising: a samplefrom a culture of at least 95% pure, stable, homogeneous post-mitotichuman neurons; and a pharmaceutically acceptable medium.
 7. Thepharmaceutical composition of claim 6 wherein said neurons aredifferentiated NT2N cells.
 8. The pharmaceutical composition of claim 6wherein said neurons are transfected, differentiated NT2N cells thathave been transfected with exogenous genetic material; whereintransfected, differentiated cells express coding sequences of saidexogenous genetic material to produce a protein product.
 9. Thepharmaceutical composition of claim 6 wherein said exogenous geneticmaterial includes nucleic acid sequences that encode a protein selectedfrom the group consisting of neurotransmitters (e.g., tyrosinehydroxlase) and neurotrophic substances such as nerve growth factor,brain-derived neurotrophic factor (BDGF), basic fibroblast growth factor(bFGF) and glial-derived growth factor.
 10. A method of generating anon-human animal model for a central nervous system disease or disordercomprising the step of: implanting a sample from a culture of at least95% pure, stable, homogeneous post-mitotic human neurons into saidnon-human animal's brain.
 11. The method of claim 10 wherein saidneurons are NT2N cells.
 12. The method of claim 10 wherein said neuronsare NT2N cells that have been transfected with exogenous geneticmaterial, wherein transfected cells express coding sequences of saidexogenous genetic material to produce a protein product.
 13. The methodof claim 10 wherein said exogenous genetic material includes nucleicacid sequences that encode proteins selected from the group consistingof normal and mutated amyloid precursor, kinases, phosphotases, normaland mutated superoxidedismutase, neurofilament proteins andapolipoprotein
 4. 14. The method of claim 10 wherein said disease ordisorder is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Huntington's disease, stroke and nerve injuries.15. The method of claim 10 wherein said animal is a rodent.
 16. Themethod of claim 10 wherein said animal is an immunocompetent rodent andfurther comprising the step of administering Cyclosporin to said rodentbefore, during and/or after implanting said cells.
 17. An non-humananimal comprising: a sample from a culture of at least 95% pure, stable,homogeneous post-mitotic human neurons implanted in the brain of saidnon-human animal.
 18. The non-human animal of claim 17 wherein saidneurons are NT2N cells.
 19. The non-human animal of claim 17 whereinsaid neurons are NT2N cells that have been transfected with exogenousgenetic material, wherein transfected cells express coding sequences ofsaid exogenous genetic material to produce a protein product.
 20. Thenon-human animal of claim 19 wherein said exogenous genetic materialincludes nucleic acid sequences that encode proteins selected from thegroup consisting of normal and mutated amyloid precursor, kinases,phosphotases, normal and mutated superoxidedismutase, neurofilamentproteins and apolipoprotein
 4. 21. A method of treating an individualsuspected of suffering from an injury, disease or disorder characterizedby nerve damage comprising the step of: implanting a sample from aculture of at least 95% pure, stable, homogeneous post-mitotic humanneurons at or near a sit of said nerve damage
 22. The method of claim 21wherein said neurons are differentiated NT2N cells.
 23. The method ofclaim 21 wherein said injury is a spinal injury and said sample isimplanted in said individual's spinal column.
 24. The method of claim 1wherein said injury is to a motor neuron.